Aconitase catalyzes the interconversion of the three tricarboxylic acids of the Krebs Cycle, citric acid, cis-aconitic acid and isocitric acid. This enzyme has long been known to require iron and sulfide ions for activity. Recently aconitase has been demonstrated to be the high potential iron-sulfur protein from mitochrondria. This project will examine the primary and tertiary structure of aconitase. First, the amino acid sequence of aconitase will be determined. Sequencing methods currently in use are expected to be adequate for solution of this part of the project. Second, chemical modifications will be initiated as a means of identifying those amino acid residues of aconitase which participate in its catalytic function and those that serve as ligands to the iron(s). Initially, these modification studies will concentrate on identifying the cysteine and arginine residues already known to react with specific reagents. Third, we will continue efforts to grow crystals of aconitase suitable for X-ray analysis. If suitable crystals can be grown, we will proceed to determine the tertiary structure of aconitase. This project is aimed at providing a sound structural basis for determining the mechanism of this important Krebs cycle enzyme and for understanding its nature as a high potential iron-sulfur protein.